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Ganglioside GM1 Contributes to the State of Insulin Resistance in Senescent Human Arterial Endothelial Cells*

  • Norihiko Sasaki
    Affiliations
    Research Team for Geriatric Medicine (Vascular Medicine), Tokyo Metropolitan Institute of Gerontology, Sakaecho 35-2, Itabashi-ku, Tokyo 173-0015, Japan
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  • Yoko Itakura
    Affiliations
    Research Team for Geriatric Medicine (Vascular Medicine), Tokyo Metropolitan Institute of Gerontology, Sakaecho 35-2, Itabashi-ku, Tokyo 173-0015, Japan
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  • Masashi Toyoda
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-3-3964-3241, ext. 4421; Fax: 81-3-3579-4776.
    Affiliations
    Research Team for Geriatric Medicine (Vascular Medicine), Tokyo Metropolitan Institute of Gerontology, Sakaecho 35-2, Itabashi-ku, Tokyo 173-0015, Japan
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  • Author Footnotes
    * This work was supported by Japan Society for the Promotion of Science KAKENHI Grant 24890298, grant-in-aid for young scientists (start-up), grant-in-aid for scientific research (C), and grant-in-aid for challenging exploratory research, and Terumo Life Science Foundation (2013 general subsidy). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:September 02, 2015DOI:https://doi.org/10.1074/jbc.M115.684274
      Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people.

      Introduction

      Vascular endothelial cells (ECs)
      The abbreviations used are: EC
      endothelial cell
      eNOS
      endothelial NO synthase
      GSLs
      glycosphingolipids
      GM3
      monosialodihexosylganglioside
      GM1
      monosialotetrahexosylganglioside
      HAEC
      human aortic endothelial cell
      EGM-2
      endothelial growth medium-2
      PDL
      population doubling level
      AMP-dNM
      N-(5′-adamantane-1′-yl-methoxy)-pentyl-1-deoxynojirimycin
      SA-β-Gal
      senescence-associated β-galactosidase
      GM2
      monosialotrihexosylceramide
      GD1a
      disialogangliotetraosylceramide
      IR
      insulin receptor
      IRS
      IR substrate
      MFI
      mean fluorescent intensity.
      constitute the endothelium of blood vessels, which forms an interface between the blood and the vessel wall and plays important roles in vascular homeostatic functions. Excessive activation or dysfunction of ECs is considered to lead to the development of vascular-related diseases, including restenosis, arteriosclerosis, and cancer (
      • Rajendran P.
      • Rengarajan T.
      • Thangavel J.
      • Nishigaki Y.
      • Sakthisekaran D.
      • Sethi G.
      • Nishigaki I.
      The vascular endothelium and human diseases.
      ). Insulin signaling regulates important functions in ECs and contributes to the maintenance of vascular integrity. For example, insulin signaling in ECs modulates NO production by endothelial NO synthase (eNOS) activation and the expression of adhesion molecules, and it also attenuates the progression of atherosclerosis (
      • Ma H.
      • Zhang H.F.
      • Yu L.
      • Zhang Q.J.
      • Li J.
      • Huo J.H.
      • Li X.
      • Guo W.Y.
      • Wang H.C.
      • Gao F.
      Vasculoprotective effect of insulin in the ischemic/reperfused canine heart: role of Akt-stimulated NO production.
      ,
      • Rask-Madsen C.
      • Li Q.
      • Freund B.
      • Feather D.
      • Abramov R.
      • Wu I.H.
      • Chen K.
      • Yamamoto-Hiraoka J.
      • Goldenbogen J.
      • Sotiropoulos K.B.
      • Clermont A.
      • Geraldes P.
      • Dall'Osso C.
      • Wagers A.J.
      • Huang P.L.
      • et al.
      Loss of insulin signaling in vascular endothelial cells accelerates atherosclerosis in apolipoprotein E null mice.
      ). So, insulin resistance in ECs, which is a dysfunction characterized by the impairment of insulin signaling, is considered to lead to the initiation and progression of vascular and vascular-related diseases (
      • Bornfeldt K.E.
      • Tabas I.
      Insulin resistance, hyperglycemia, and atherosclerosis.
      ,
      • Kubota T.
      • Kubota N.
      • Kumagai H.
      • Yamaguchi S.
      • Kozono H.
      • Takahashi T.
      • Inoue M.
      • Itoh S.
      • Takamoto I.
      • Sasako T.
      • Kumagai K.
      • Kawai T.
      • Hashimoto S.
      • Kobayashi T.
      • Sato M.
      • et al.
      Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle.
      ).
      The aging of the population worldwide is resulting in increasing numbers of older people, among whom vascular disease is the leading cause of death. Senescence and aging of ECs have been considered to increase the risk of vascular diseases (
      • Minamino T.
      • Komuro I.
      Vascular cell senescence: contribution to atherosclerosis.
      ,
      • Favero G.
      • Paganelli C.
      • Buffoli B.
      • Rodella L.F.
      • Rezzani R.
      Endothelium and its alterations in cardiovascular diseases: life style intervention.
      ). Therefore, it is important to clarify the mechanisms underlying senescence and aging-associated diseases to lower the risk for vascular disease and extend healthy life expectancy. From previous research, it is known that senescence and aging cause EC dysfunctions such as reduced NO production and elevated inflammation (
      • Wang J.C.
      • Bennett M.
      Aging and atherosclerosis: mechanisms, functional consequences, and potential therapeutics for cellular senescence.
      ). Consequently, it has been speculated that senescence and aging produce insulin resistance in ECs, but until now, the molecular mechanisms of the insulin resistance that occurs with senescence and aging have been unclear. To investigate these mechanisms, we focused on glycosphingolipids (GSLs).
      GSLs are composed of a glycan structure attached to a lipid tail containing the sphingolipid ceramide. GSLs are widely expressed on cell membranes in lower and higher eukaryotic organisms. GSLs have frequently been used as important developmental marker molecules and have been suggested to have important biological functions (
      • Varki A.
      • Cummings R.D.
      • Esko J.D.
      • Freeze H.H.
      • Stanley P.
      • Bertozzi C.R.
      • Gerald W.H.
      • Marilynn E.E.
      Essentials of Glycobiology.
      ,
      • Lingwood C.A.
      Glycosphingolipid functions.
      ). So far, studies on gangliosides (molecules composed of GSLs with one or more sialic acids) related to senescence and aging have been reported in neural tissues but not yet in ECs (
      • Ohsawa T.
      Changes of mouse brain gangliosides during aging from young adult until senescence.
      ,
      • Jiang L.
      • Bechtel M.D.
      • Bean J.L.
      • Winefield R.
      • Williams T.D.
      • Zaidi A.
      • Michaelis E.K.
      • Michaelis M.L.
      Effects of gangliosides on the activity of the plasma membrane Ca2+-ATPase.
      ). It has been demonstrated that gangliosides are fine regulators of receptor tyrosine kinases signaling, including insulin signaling, and that changing cell surface ganglioside compositions in physiopathological conditions results in altered cellular responses (
      • Bremer E.G.
      • Hakomori S.
      Gangliosides as receptor modulators.
      ,
      • Hakomori S.
      • Igarashi Y.
      Functional role of glycosphingolipids in cell recognition and signaling.
      ). In 3T3-L1 adipocytes, the monosialodihexosylganglioside (GM3) was found to contribute to insulin resistance in pathological conditions such as obesity (
      • Tagami S.
      • Inokuchi Ji J.
      • Kabayama K.
      • Yoshimura H.
      • Kitamura F.
      • Uemura S.
      • Ogawa C.
      • Ishii A.
      • Saito M.
      • Ohtsuka Y.
      • Sakaue S.
      • Igarashi Y.
      Ganglioside GM3 participates in the pathological conditions of insulin resistance.
      ). Furthermore, it was shown that potent inhibitors of GSLs improve ganglioside-mediated insulin sensitivity in pathological model mice (
      • Aerts J.M.
      • Ottenhoff R.
      • Powlson A.S.
      • Grefhorst A.
      • van Eijk M.
      • Dubbelhuis P.F.
      • Aten J.
      • Kuipers F.
      • Serlie M.J.
      • Wennekes T.
      • Sethi J.K.
      • O'Rahilly S.
      • Overkleeft H.S.
      Pharmacological inhibition of glucosylceramide synthase enhances insulin sensitivity.
      ,
      • Zhao H.
      • Przybylska M.
      • Wu I.H.
      • Zhang J.
      • Siegel C.
      • Komarnitsky S.
      • Yew N.S.
      • Cheng S.H.
      Inhibiting glycosphingolipid synthesis improves glycemic control and insulin sensitivity in animal models of type 2 diabetes.
      ). Therefore, it is considered that gangliosides play important roles in insulin resistance. However, the remaining issues are as follows: (i) whether gangliosides contribute to insulin resistance in ECs and (ii) what kinds of gangliosides contribute to the dysfunction.
      We focused on gangliosides of ECs, and we hypothesized that changes in the abundance of cell surface gangliosides with senescence and aging contribute to EC dysfunctions such as insulin resistance. In this study, we revealed for the first time that monosialotetrahexosylganglioside (GM1) among the several gangliosides species was increased in abundance on the cell surface of ECs with cellular senescence (replicative and premature) and aging and that the increased abundance of GM1 resulted in a state of insulin resistance in senescent ECs.

      Discussion

      Insulin signaling in ECs plays important roles in the maintenance of vascular integrity. Therefore, the development of insulin resistance in ECs leads to increased vascular morbidity and mortality. The remaining issues are the molecular mechanisms of the insulin resistance that occurs with senescence and aging. In this study, we have demonstrated that the abundance of GM1 increases on the cell surface of senescent and aged ECs and contributes to insulin resistance in senescent ECs. Therefore, we propose that the increased abundance of GM1 in ECs with senescence and aging is a risk factor for vascular diseases in older people.
      GM1 is synthesized by glycosyltransferases and sialidase (NEU3) as shown in Fig. 1B. The changed expression of these enzymes leads to an increased production of GM1. In fact, the overexpression of B4GALNT1 and B3GALT4 or NEU3 in mammalian cells was reported to induce an increase in the abundance of GM1 (
      • Sasaki A.
      • Hata K.
      • Suzuki S.
      • Sawada M.
      • Wada T.
      • Yamaguchi K.
      • Obinata M.
      • Tateno H.
      • Suzuki H.
      • Miyagi T.
      Overexpression of plasma membrane-associated sialidase attenuates insulin signaling in transgenic mice.
      ,
      • Mitsuda T.
      • Furukawa K.
      • Fukumoto S.
      • Miyazaki H.
      • Urano T.
      • Furukawa K.
      Overexpression of ganglioside GM1 results in the dispersion of platelet-derived growth factor receptor from glycolipid-enriched microdomains and in the suppression of cell growth signals.
      ). In senescent ECs, we found that the expression of B4GALNT1, which catalyzes the synthesis of GM2 (a GM1 precursor), was up-regulated. Furthermore, a decrease in the abundance of GD3 was observed in senescent ECs, suggesting that the GM2 and GM1 synthetic pathway is predominant in senescence. These results are suggested to be one explanation for the increased abundance of GM1 in senescent cells. In aged ECs (HAECs-elder), an increased abundance of GM1 (relative mean fluorescent intensity (MFI) ∼500) was detected, compared with that in senescent HAECs-middle (relative MFI ∼200). Also, three glycosyltransferases involved in GM1 synthetic pathways, ST3GAL5, B4GALNT1, and B3GALT4, were up-regulated in the aged ECs. Aging may be accompanied by altered expression levels of these enzymes. Further study should be required to clarify the mechanisms regulating the expression of these enzymes and to identify the key regulators of GM1 abundance in ECs with senescence and aging.
      As shown in Fig. 3, D and H, the expression levels of IR on the cell surface were slightly reduced in senescent ECs. This reduction in IR expression was restored after AMP-dNM treatment, which lowered the increased GM1 levels in senescent ECs (data not shown). Furthermore, exogenous GM1 treatment for 3 days induced a slight reduction in the expression of IR on the cell surface of ECs, which was similar to that observed in senescent ECs (data not shown). It has been reported that the cell surface abundances of gangliosides affect the expression levels of raft-associated proteins (
      • Furukawa K.
      • Ohmi Y.
      • Ohkawa Y.
      • Tokuda N.
      • Kondo Y.
      • Tajima O.
      • Furukawa K.
      Regulatory mechanisms of nervous systems with glycosphingolipids.
      ). Thus, it is suggested that increased GM1 may affect the expression of IR on the cell surface, although the molecular mechanism underlying the regulation of cell surface IR via GM1 is unknown. In contrast, 24 h after treatment of ECs with exogenous GM1, insulin resistance was induced without any reduction in the expression of IR on the cell surface (Fig. 6C). Therefore, this result indicates that a slight reduction in the expression of IR on the cell surface does not affect insulin signaling in senescent ECs and confirms that an increased abundance of GM1 functionally contributes to insulin resistance with senescence.
      In this study, we have demonstrated that increased GM1 contributes to insulin resistance in senescent ECs, presumably due to a physical association between GM1 and IR. It has not been established how increased GM1 regulates insulin signaling in ECs, but we propose two possible mechanisms (Fig. 8). One is that an association between the GM1 carbohydrate moiety and an epitope in the extracellular domain of IR leads to the exclusion of IR from caveolae, as was recently found for ganglioside GM3 and IR (
      • Kabayama K.
      • Sato T.
      • Saito K.
      • Loberto N.
      • Prinetti A.
      • Sonnino S.
      • Kinjo M.
      • Igarashi Y.
      • Inokuchi J.
      Dissociation of the insulin receptor and caveolin-1 complex by ganglioside GM3 in the state of insulin resistance.
      ). The second proposed mechanism is that an increased abundance of GM1 affects the catalytic activity of IR and attenuates signal transduction, as has been suggested by previous reports (
      • Sasaki A.
      • Hata K.
      • Suzuki S.
      • Sawada M.
      • Wada T.
      • Yamaguchi K.
      • Obinata M.
      • Tateno H.
      • Suzuki H.
      • Miyagi T.
      Overexpression of plasma membrane-associated sialidase attenuates insulin signaling in transgenic mice.
      ). To clarify the molecular mechanism underlying the development of insulin resistance via an increased abundance of GM1 in ECs, further study is required.
      Figure thumbnail gr8
      FIGURE 8Two proposed mechanisms to explain how an increased abundance of GM1 can increase insulin resistance in senescent ECs. 1) exclusion of IR from caveolae; GM1 interacts with IR followed by the dissociation of IR from caveolae, resulting in insulin resistance; 2) reduction of IR catalytic activity; increased GM1 interacts with IR and then directly affects IR kinase activity, resulting in insulin resistance.
      In adipocytes, TNFα treatment was reported to induce an increased abundance of GM3 in the cell membrane, and this increased abundance of GM3 was found to produce insulin resistance (
      • Tagami S.
      • Inokuchi Ji J.
      • Kabayama K.
      • Yoshimura H.
      • Kitamura F.
      • Uemura S.
      • Ogawa C.
      • Ishii A.
      • Saito M.
      • Ohtsuka Y.
      • Sakaue S.
      • Igarashi Y.
      Ganglioside GM3 participates in the pathological conditions of insulin resistance.
      ). In this study, we have demonstrated that an increased abundance of GM1 produces insulin resistance in ECs. We also investigated the abundances of gangliosides in ECs after TNFα treatment. In ECs, GM1 was increased but GM3 was not.
      N. Sasaki, Y. Itakura, and N. Toyoda, unpublished data.
      Furthermore, in transgenic mice showing insulin resistance, NEU3 overexpression induced increases in the abundances of GM1 and GM2 in several tissues, including liver (
      • Sasaki A.
      • Hata K.
      • Suzuki S.
      • Sawada M.
      • Wada T.
      • Yamaguchi K.
      • Obinata M.
      • Tateno H.
      • Suzuki H.
      • Miyagi T.
      Overexpression of plasma membrane-associated sialidase attenuates insulin signaling in transgenic mice.
      ). Thus, it is possible that the abundances of gangliosides related to insulin resistance differ among cell types and tissues. So, clarifying the significance of the abundance of each ganglioside in relation to tissue-specific insulin resistance could lead to a deeper understanding of each pathological condition and thus to more efficient drug discovery for the treatment of insulin resistance-related diseases.
      Beneficial effects of AMP-dNM on pathological model mice have been reported. AMP-dNM treatment restores insulin sensitivity in ob/ob mice (
      • Aerts J.M.
      • Ottenhoff R.
      • Powlson A.S.
      • Grefhorst A.
      • van Eijk M.
      • Dubbelhuis P.F.
      • Aten J.
      • Kuipers F.
      • Serlie M.J.
      • Wennekes T.
      • Sethi J.K.
      • O'Rahilly S.
      • Overkleeft H.S.
      Pharmacological inhibition of glucosylceramide synthase enhances insulin sensitivity.
      ) and also inhibits atherosclerosis in APOE*3 Leiden as well as low-density lipoprotein receptor−/− mice (
      • Bietrix F.
      • Lombardo E.
      • van Roomen C.P.
      • Ottenhoff R.
      • Vos M.
      • Rensen P.C.
      • Verhoeven A.J.
      • Aerts J.M.
      • Groen A.K.
      Inhibition of glycosphingolipid synthesis induces a profound reduction of plasma cholesterol and inhibits atherosclerosis development in APOE*3 Leiden and low-density lipoprotein receptor−/− mice.
      ). In the former report (
      • Aerts J.M.
      • Ottenhoff R.
      • Powlson A.S.
      • Grefhorst A.
      • van Eijk M.
      • Dubbelhuis P.F.
      • Aten J.
      • Kuipers F.
      • Serlie M.J.
      • Wennekes T.
      • Sethi J.K.
      • O'Rahilly S.
      • Overkleeft H.S.
      Pharmacological inhibition of glucosylceramide synthase enhances insulin sensitivity.
      ), it was suggested that reducing the increased abundance of GM3 in adipocytes by AMP-dNM treatment improves insulin sensitivity. In the latter report (
      • Bietrix F.
      • Lombardo E.
      • van Roomen C.P.
      • Ottenhoff R.
      • Vos M.
      • Rensen P.C.
      • Verhoeven A.J.
      • Aerts J.M.
      • Groen A.K.
      Inhibition of glycosphingolipid synthesis induces a profound reduction of plasma cholesterol and inhibits atherosclerosis development in APOE*3 Leiden and low-density lipoprotein receptor−/− mice.
      ), lowering of plasma cholesterol by AMP-dNM treatment was proposed to reduce the development of atherosclerosis. Recently, it has been demonstrated that insulin resistance in ECs plays major roles in type 2 diabetes and cardiovascular diseases (
      • Bornfeldt K.E.
      • Tabas I.
      Insulin resistance, hyperglycemia, and atherosclerosis.
      ,
      • Kubota T.
      • Kubota N.
      • Kumagai H.
      • Yamaguchi S.
      • Kozono H.
      • Takahashi T.
      • Inoue M.
      • Itoh S.
      • Takamoto I.
      • Sasako T.
      • Kumagai K.
      • Kawai T.
      • Hashimoto S.
      • Kobayashi T.
      • Sato M.
      • et al.
      Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle.
      ). In this study, we have demonstrated that increased GM1 contributes to insulin resistance in ECs. It is considered that an increased abundance of GM1 on ECs occurs in pathological conditions such as obesity and atherosclerosis, and it has been reported that senescent ECs are present in atherosclerotic lesions (
      • Minamino T.
      • Miyauchi H.
      • Yoshida T.
      • Ishida Y.
      • Yoshida H.
      • Komuro I.
      Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction.
      ). Therefore, we speculate that the reduction of increased GM1 abundance by AMP-dNM treatment could also contribute to the improvement of insulin resistance-related pathological conditions.
      Increased GM1 is known to affect the cell surface expression of raft-associated proteins and to contribute to the reduction of membrane fluidity (
      • Mitsuda T.
      • Furukawa K.
      • Fukumoto S.
      • Miyazaki H.
      • Urano T.
      • Furukawa K.
      Overexpression of ganglioside GM1 results in the dispersion of platelet-derived growth factor receptor from glycolipid-enriched microdomains and in the suppression of cell growth signals.
      ,
      • Nishio M.
      • Fukumoto S.
      • Furukawa K.
      • Ichimura A.
      • Miyazaki H.
      • Kusunoki S.
      • Urano T.
      • Furukawa K.
      Overexpressed GM1 suppresses nerve growth factor (NGF) signals by modulating the intracellular localization of NGF receptors and membrane fluidity in PC12 cells.
      ,
      • Dong Y.
      • Ikeda K.
      • Hamamura K.
      • Zhang Q.
      • Kondo Y.
      • Matsumoto Y.
      • Ohmi Y.
      • Yamauchi Y.
      • Furukawa K.
      • Taguchi R.
      • Furukawa K.
      GM1/GD1b/GA1 synthase expression results in the reduced cancer phenotypes with modulation of composition and raft-localization of gangliosides in a melanoma cell line.
      ). Several cellular dysfunctions, such as impaired signal transduction, etc., are considered to be due to changes in the composition of gangliosides in the cell membrane. In this study, we have shown that the abundance of GM1 is increased on senescent ECs, resulting in insulin resistance. Furthermore, it is possible that an increased abundance of GM1 on ECs with senescence may also cause other EC dysfunctions, although further studies are required to investigate this.
      Taken together, as described above, we have shown that the abundance of GM1 on the surface of ECs is increased with senescence and aging. We suggest that an increased abundance of GM1 in cell membranes may produce insulin resistance throughout the body, resulting in the initiation and/or progression of serious vascular diseases. Notably, an increased abundance of GM1 with senescence and aging may contribute to the elevated risk of vascular diseases in older people. We propose that GM1 could be an attractive target for the detection, prevention, and treatment of insulin resistance, particularly in older people. The inhibition of GM1 synthesis and the interaction between GM1 and IR may offer avenues for the future development of new therapeutic strategies. To facilitate the clinical translation of these findings, further study will be required to identify the key regulators of GM1 homeostasis and clarify the molecular mechanism underlying the production of insulin resistance via an increased abundance of GM1.

      Author Contributions

      N. S. designed and performed experiments, analyzed data, and wrote all of the paper. Y. I. analyzed data. M. T. designed experiments and wrote the paper with N. S. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgment

      We thank Noriko Gojo for help with the cell culture.

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